CN1376158A - Oligosaccharides derived from ribose-ribitol-phosphate, and vaccines containing them - Google Patents
Oligosaccharides derived from ribose-ribitol-phosphate, and vaccines containing them Download PDFInfo
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- CN1376158A CN1376158A CN00813473A CN00813473A CN1376158A CN 1376158 A CN1376158 A CN 1376158A CN 00813473 A CN00813473 A CN 00813473A CN 00813473 A CN00813473 A CN 00813473A CN 1376158 A CN1376158 A CN 1376158A
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- ribitol
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/102—Pasteurellales, e.g. Actinobacillus, Pasteurella; Haemophilus
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/02—Acyclic radicals, not substituted by cyclic structures
- C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/02—Acyclic radicals, not substituted by cyclic structures
- C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
- C07H15/08—Polyoxyalkylene derivatives
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/18—Acyclic radicals, substituted by carbocyclic rings
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Abstract
The present invention relates to in particular with the chemical synthesis of oligosaccharide mixtures derived of ribose-ribitol-phosphate, which are used as active principle in vaccines for the prevention of infections caused by Haemophilus influenzae type b (Hib), as well as with the vaccines containing said oligosaccharide mixtures. The oligasaccharide mixtures obtained by chemical synthesis of the present invention, comprise repeating units of formulae (phosphate-ribosa-ribitol)n or (ribose-ribitol-phosphate)n of at least 5 compounds of structure A or B, which represent the repeating unit of the capsular polysaccharide of Haemophilus influenzae type b and differ only by n, being n a value contained between 4 and 25 (n>=4 y<=25), and wherein R1 or R2 is a spacer for conjugation to a carrier, with the condition of R1=spacer if R2=H, or R2=spacer if R1=H.The invention also is related with the immunogens containing such oligosaccharide mixtures, with the vaccines containing said immunogens and with the methods to prepare these oligosaccharides as mixtures. Furthermore, the invention includes the use of the vaccines, alone or combined with other vaccines, for the prevention of the infections caused by Haemophilus influenzae type b.
Description
The technology part
The present invention relates to field of medicaments, relate in particular to the chemosynthesis of ribose-ribitol-phosphoric acid deutero-oligosaccharide mixture, and relate to the vaccine that contains this oligosaccharide mixture, wherein said oligosaccharide mixture is used as the activeconstituents in the vaccine, is used to prevent the infection that is caused by Haemophilus influenzae (Haemophilus influenzae) b type (Hib).
Prior art
Worldwide, human health in the serious threat of Haemophilus influenzae b type.This bacterium mainly causes meningitis, pneumonia, meeting pharyngitis and other respiratory tract disease to children below 5 years old.Observed consequence is: in many countries, the problem from sense of hearing problem to severe mental retardation is arranged more than 30% among this disease survivor.The nearest estimation of the World Health Organization shows that the annual in the world children above 550000 die from the disease that is caused by Haemophilus influenzae b type.
The Haemophilus influenzae capsular polysaccharide of purifying can be induced protective immunity in the adult, but children are then induced very low immune response, and particularly the baby below 2 years old does not have immune response to it.
Capsular polysaccharide has following structure:
Shown that subject matter is antigenic self-characteristic,, be T cell dependence antigen not, the immunity system that can not stimulate the baby of prematurity still because it is a kind of polysaccharide.Shown that this problem can be by solving polysaccharide and the protein that is known as carrier covalently bound (combination).The product that obtains like this is known as combined vaccine, and it can induce 2 months big babies to produce the antibody of protectiveness level.
People such as Chu, (infection immunity (Infection immunity) 1983,40 245-256) with the Toxoid,tetanus coupling of natural capsular polysaccharide and cyanogen bromide-activated, has obtained binding substances.
Gordon (U.S. Patent number 4,496,538) is with cyanogen bromide-activated natural polysaccharide, then with it by adipic acid dihydrazide and diphtheria toxoid coupling.
People such as Hilman (U.S. Patent number 4,459,286) will be behind initiate activation natural polysaccharide by 6-aminocaprolc acid and meningococcal outer membrane protein coupling.
In combining method on all, covalently bound is to carry out between several groups of capsular polysaccharide and carrier protein.
Induce the ability of protective immunity to depend on the structure of conjugate to the baby.When use polysaccharide when carrying out coupling because the group that participates in connecting is randomly dispersed on the polysaccharide chain, thereby make the sign of each batch become very complicated.
Is very difficult by physico-chemical process to all these vaccine analyses; Therefore ordinary method is by the immunogenicity of research to laboratory animal, thereby each batch estimated.But conjugate is different with it to the behavior of laboratory animal to children.
According to the stabilised quality standard of The World Health Organization (WHO) (M.R.Holliday and C.Jones, biology (Biologicals), 1999,27,51-53), the control of coupling vaccine should based on use physico-chemical process illustrate batch with criticize between similarity.
In order to promote this work, more should on molecular level, define the coupling vaccine.The other method that addresses this problem is synthetic capsular polysaccharide fragment.Make up the antigenic method of Haemophilus influenzae b type two step key steps arranged by synthesizing: synthetic disaccharides intermediate with and oligomerization.Some approach have been synthesized after deliberation at this.
People such as Beuvery (European patent application EP O0276516; United States Patent (USP) 5,034,519; Tetrahedron wall bulletin (Tetrahedron Lett.) 28,1553,1987) and people's (carbohydrate chemistry magazine (J.Carbohydr.Chem) such as Hoogerhout, 7,1988,399-416) obtain by synthetic capsular polysaccharide fragment, the capsular polysaccharide fragment of wherein applying for a patent contains 3 to 20 repeating units.For reaching this purpose, at first prepare disaccharides intermediate 2, synthetic by solid state chemistry or solution then, preparation contains the oligomer (people such as Elie, Rec.Trav.Chim.Pays-Bas 108,1989,219) of 6 repeating units.Oligomer combines with protein or peptide by spacerarm.On one's body mouse, the conjugate tripolymer is the same with the commercially available vaccine by the capsular polysaccharide preparation, has immunogenicity.
In a preferred embodiment, set forth following route of synthesis, used following strategy: 1-synthetic kernel sugar alcohol, 2-and ribose coupling, 3-imports substituting group to the ribose units selectivity, and 4-imports the phosphorus activating group.Use this approach, can only obtain two crucial sugar derivativess 2 after 15 reactions steps.
Overall yield is that 7% (people such as Hermans, Recl.Trav.Chim.Pays-Bas 1987,106,498-504).Further, this method has two main weak points: this method comprises the chromatography step 11 times; And for the processing of oligomerization, the blocking group of its crucial intermediate is undesirable.
When oligomerization, or synthetic second step, method therefor is synthetic for the solution that is undertaken by the activation phosphotriester, makes each round-robin productive rate based on disaccharides between 70-90%.The main weak point of this type of step is: can not prepare the fragment that contains more than 4 repeating units, because its productive rate descends fast.Disaccharides intermediate 2 has three different blocking groups, this make end product go the protection very the difficulty.Therefore this disaccharides is not easy to carry out the oligomerization effect with mechanochemical method.Because event has been reported in synthesizing of six aggressiveness.
Only (people such as Peters CCAM, infection immunity (Infect.Immunity) 1991,59 3504-10) have been reported the coupling of tripolymer and Toxoid,tetanus and to the immunogenicity of mouse and monkey in immunoassay one literary composition.
G.Just, J.Upeslacis (European patent application EP 0320942, L.Chan; G.Just, tetrahedron (Tetrahedron), 46,1990,151-162) also synthesized the capsular polysaccharide fragment, and be synthetic in solution chemistry by the disaccharides intermediate.In order to prepare best antigen synthetic intermediate, selected different approach: 1-synthetic kernel sugar alcohol, 2-is synthetic to have the ribose units of the group that adequately protects, 3-coupling ribose and ribitol, and 4-imports the phosphorus mobilizing function.
In this way, intermediate 3 is prepared as phosphoramidate (phosphoramidite), it shows the better selectivity to blocking group when oligomerization is handled.For reaching this purpose, need more step.Can obtain crucial derivative in the 19th step, and use 8 chromatography steps to carry out purifying.
Make disaccharides 3 oligomerizes in solution, obtain containing the capsular polysaccharide fragment of 3 repeating units, its productive rate based on disaccharides is between each circulation 70-90%.
People such as Kandil (synthetic wall bulletin (Syn.Lett.), 1992,555-7), people such as Chon (PCT patent application WO93/15205 and United States Patent (USP) 5,679,352) are with same disaccharides intermediate 3 and by mechanochemical method, the fragment of having synthesized capsular polysaccharide.As upholder, the fragment of acquisition contains 6 repeating units of as many as with the mono methoxy macrogol, and each round-robin productive rate is 95%.
(carbohydrate chemistry magazine 10,1991 1-22) with similar disaccharides intermediate and mechanochemical method, has obtained the fragment of 10 repeating units to people such as people such as Krivan (PCT patent application WO94/00149) and Nilsson.This fragment is by spacerarm and the coupling of Hib adhesin.With 21 the step obtained this phosphonic acid ester intermediate, overall yield is 5%.Also need the chromatography step at least 7 times.Carry out oligomerization with the H-phosphonic acid ester and handle, and carry out mechanochemical method with Merrifield ammonification resin.Can obtain the antigen that each circulation 97-99% mixes.
People such as Chiu Machado (the carbohydrate chemistry magazine, 13 1994,465-474) reported by the synthetic effective procedure suitable, protected ribose derivates of glucose with Cuba's patent 22424.They use this derivative, have prepared crucial disaccharides in 20 reactions steps.
Make the synthetic segmental preparation of Hib that is applied to, and be the synthetic of disaccharides intermediate as one of aspects of vaccine difficulty them.
All methods of listing have above reflected the current state of the art of modern carbohydrate chemistry, yet there are two main serious deficiencies technically in they.In synthetic, repeatedly use chromatographic step, thereby it is impracticable that this is synthesized on technical scale, and synthesis step is often more.Be in ribose units, to have imported benzyl with the subject matter in the synthetic disaccharides of less reactions steps.
The oligomerization of disaccharides intermediate is handled and can be carried out in solution, but extremely inconvenient, accessible overall dimension is limited to 3-4 repeating unit.Also can be undertaken by mechanochemical method.Mechanochemical method can make the oligosaccharides of preparation contain 6 to 10 repeating units, and each circulation high yield.But usually two serious problems that exist are: actual yield has only 10-15% because in each circulation in order to obtain the disaccharides intermediate that height mixes, often need high excessive disaccharides, between 3 to 10 molar equivalents.Excessive disaccharides is loose in treating processes.Another problem is: need two kinds of different derivatives usually, one is used for and the solid support coupling, second extension that is used for chain.
Synthetic single, pure oligose fragment is the synthetic antigenic common point of Hib of above-mentioned report, is one of their main purpose simultaneously, and wherein said oligose fragment is not only in structure, and on chain length equal indifferences.All these are based on following supposition: single molecular antigen is essential to the anti-Hib combined vaccine that obtains than stabilised quality, because it is more easy to control.
Developed novel method, also developed the novel method of one of terminal position by them activation oligosaccharide mixture with natural polysaccharide produced in fragments oligose fragment.
United States Patent (USP) 4,808,700, United States Patent (USP) 4,761,283 and compounding sugar magazine (Glycoconjugate J.), 1989,6, among 489-498 people such as () R.C.Seid, with peryodate oxidation natural polysaccharide and the resulting fragment of purifying.The mixture of oligose fragment is activated by two terminal positions, shown in following scheme.These oligosaccharides combine with CRM197 by the reductibility aminating reaction.This point can find out from this scheme that two coupling sites are different.In case carry out coupling, have two binding oligosaccharide families of tool different structure at least.On the other hand, the per-cent of oligosaccharides can not determine two different loci of same oligosaccharides and same protein well, or with the result that is connected of two different proteins molecules.All these phenomenons can produce heterogeneity, and make control become difficult.
On the other hand, at United States Patent (USP) 5,153,312 and vaccine (Vaccine) 1999,17, among the 1251-63 (people such as P.Constantino), reported and used the acetolysis natural polysaccharide, and the resulting oligose fragment mixture of purifying.Product activates by a series of reaction, and wherein spacerarm imports by the reductibility ammonification with 1 at high pH and can influence under the temperature of integrity of oligosaccharide mixture.On the other hand, a part of antigen its carbonyl hemiacetal through the reduction after, possible inactivation, this point is shown in following scheme.Further, by one of hexanodioic acid terminal ester functional group, the oligosaccharides amino derivative is replaced by the Acibenzolar selectivity of hexanodioic acid.Remaining ester functional group still have can with protein link coupled mobilizing function,
Two kinds of vaccines that prepared by the product of natural polysaccharide fragment have been useful for the immunization to a large amount of children.This shows the coupling vaccine that can use oligosaccharides to produce anti-Hib, and fully the repeatability and the quality of control product are possible simultaneously, and wherein said oligosaccharides is not single size, but in certain magnitude range.Even the conjugate of Huo Deing is more definite by the product that the polysaccharide direct activation obtains than those by this method, but actually, to natural polysaccharide fragment fragmentation, import feature activating group subsequently, and the antigen molecule definition and the purity that reach the same level of antigen that obtains as chemosynthesis are impossible.
To the oligosaccharides that use has clear and definite size, also be to use in the control of coupling vaccine of oligosaccharide mixture of not of uniform size but all the other structure homogeneities, there is not remarkable advantages.This point has been illustrated in advanced experiment, be easy to measure the composition (people such as P.Constantino of Hib oligosaccharide mixture by state-of-the-art analytical procedure, vaccine 1999,17, people such as 1251-1263 and D.Prioeti, European sugared discussion (European CarbohydrateSymposium), Galway, in July, 1999, PB014).
On the other hand, between vaccine of forming by Hib oligosaccharide mixture not of uniform size and the vaccine formed by the oligosaccharides of single size (when fragment during) greater than three repeating units, the immunity behavior does not have difference (people such as S.Pillai, infect and immunity (Infection and Immunity), 1991,59,4371-6; People such as A.A Kandil, the compounding sugar magazine, 1997,14,13-7 and Peters CCAM wait the people, infection immunity 1991,59,3504-10).
All viewpoints are all thought: extra certain situation makes selects best single big molehill.The oligosaccharides of 4-6 repeating unit is more synthetic, and their size is enough to induce sufficient immune response usually.But the amount of the necessary carrier protein of this vaccine is very high, and the less stable of oligosaccharides in the vaccine, because the degradation process that the hydrolytic action by phosphodiester takes place is ordered about fragment very little, that be less than 4 repeating units and protein coupling very soon, and therefore inactivation.It is more stable in principle in the fragment of 8-20 repeating unit to have size, because after being degraded to their half size, remaining and carrier protein link coupled fragment is still greater than 4 repeating units.Amount to the necessary carrier protein of vaccine dose is also less.
But, be impossible with the fragment of the synthetic size of solution methods between 10-20, and this also is a challenge to solid phase method according to the prior art of Hib oligosaccharides.In fact, the oligosaccharides that does not at all have this size in the example of former report.Another disadvantage is: immunoreactive T cell dependency, and this children are reached good immune response is a very important aspect.The T cell dependency of polysaccharide with the increase of its size reduce (C.Fern á ndez, E.Sverremark, cellular immunization (Cell Immunol) 1994,153,67-78).
In a word, the antigenic method of arbitrary competitive synthetic Hib must be: can reduce the step that arrives crucial disaccharides, reduce the number of times of chromatographic step, and be increased in the output in the oligomerization treating processes significantly especially.
The oligosaccharide mixture that obtains by the hydrolysis natural polysaccharide contains the component of two kinds of size interval, and they are respectively got the chief and have reduced deficiency.If can obtain similar mixture by synthetic, it will have the advantage that contains two kinds of size interval.Because obtain by synthetic, this mixture will be more definite and purer, and contain the spacerarm of accurate ratio and exact position.
Summary of the invention
The present invention be more particularly directed to be derived from the chemosynthesis of the oligosaccharide mixture of ribose-ribitol-phosphoric acid, and the vaccine that contains this oligosaccharide mixture, wherein said oligosaccharide mixture is used as the activeconstituents in the vaccine, is used to prevent the infection that is caused by Haemophilus influenzae b type (Hib).
The present invention's the oligosaccharide mixture that obtains by chemosynthesis comprises at least 5 compounds with structure A or B, the formula of its repeating unit is (phosphoric acid-ribose-ribitol) n or (ribose-ribitol-phosphoric acid) n, it represents the repeating unit of the capsular polysaccharide of Haemophilus influenzae b type, and it is different having only n, wherein n is the value (n4 and 25) between 4 and 25, R
1Or R
2Be to be used for and carrier link coupled spacerarm, its condition is: if R
2=H, then R
1If=spacerarm is or R
1=H, then R
2=spacerarm.
The present invention also relates to contain the immunogen of this type of oligosaccharide mixture, and contain this immunogenic vaccine, prepare the method for these oligosaccharide mixtures in addition.Further, the present invention includes the purposes of vaccine, its can be separately or with other vaccine coupling, be used for preventing the infection that causes by Haemophilus influenzae b type.
Can be to obtain, can fully define the regular oligosaccharide mixture of size than efficient manner according to the present invention by chemosynthesis.This mixture has higher purity, and it obtains with better simply technological method.Discovery is fabulous with the coupling vaccine of the present invention's mixture preparation in addition, is easy to control because their production reaches.
Another object of the present invention is the synthetic method that obtains above-mentioned oligosaccharide mixture, it can be characterized by single stage method, it is controlled polycondensation between disaccharides, spacerarm and the promoter to key, and also can be according to following true the sign: antigenic mean size can be controlled by each being participated in components in proportions, their addition sequence and reaction times.
Another object of the present invention is that above-mentioned immunogen is prepared as vaccine, and the purposes of the disease that causes with opposing Haemophilus influenzae b type is wherein used or without adjuvant or other additive.
Another object of the present invention is that previously described mixture combines with other vaccine or debond prepares the purposes of combined vaccine, and wherein said other vaccine is as the vaccine of resistance of hepatitis B, DPT, meningococcemia A, B, C, anti-streptococcus pneumoniae 1,5,6B, 6A, 14,19F, 19A, 23F and poliomyelitis.
Another object of the present invention is to optimize crucial disaccharides synthetic method, and wherein said disaccharides is required for synthetic Hib oligosaccharides.Optimization comprises: found a selectivity benzyl reaction new, that be used for disaccharides 4, made it be converted into disaccharides 5, in a step benzyl protection group has been imported ribose units, entire method is obviously shortened and become simple.
Purpose of the present invention also has the step of optimizing synthetic intermediate 4, makes it can be with prepared in high purity in 11 steps, and does not use chromatography method.
Another object of the present invention is the purposes of previously described oligosaccharides, and it combines with immunologic inertia material (as polyacrylamide, polystyrene, latex), is used to resist-Hib detection of antibodies and quantitative.
Novel part of the present invention is the composition of resulting oligosaccharide mixture, it is difference with the difference of the repeating unit of the Haemophilus influenzae b type capsular polysaccharide that has spacerarm, wherein spacerarm only combines with one of its terminal position, and this position is designed when synthetic in advance.It has reacted same rule and homostyructure.This oligosaccharide mixture contains the fragment of two kinds of different size interval, mainly between 4-8 and 8-20, with the advantage that obtains every kind of interval and the shortcoming when having reduced by every group of group Individual existence.
Further, novel part of the present invention is to prepare by chemosynthesis the method for this type of mixture itself, and it makes the preparation of product have high duplication and high efficiency.Equally, by the present invention, only also set forth and just can a step benzyl protection group have been imported ribose units with a reaction, increased the efficient of crucial two sugar derivativess preparation, wherein said two sugar derivativess are used for synthetic this type of oligosaccharides.
Synthesizing from preparing derivative 5-O-allyl group-2,3 of disaccharides intermediate, 4-three-O-benzyl-D-ribitol 14 begins, and the latter prepares by 9 step chemical reactions, and it provides in following scheme.D-ribose wax is according to the aforesaid step in this area (people such as Leonart, J.Het.Chem., 1966,3,485) be converted into isopropylidene derivative 6, on the 5th,, use sulfuric acid hydrolysis isopropylidene group in methyl alcohol then with allyl bromide 98 allylation under the inversion of phases condition, so that derivative 8 to be provided, in dimethyl formamide, carry out benzylization with benzyl chloride and sodium hydride then.
With the mixture hydrolyzing methyl of acetate and hydrochloric acid, use sodium borohydride reduction then.Derivative 5-O-allyl group-2,3-two-O-benzyl-D-ribitol 11 is adsorbed on the silica gel, and by means of percolation process, uses hexanaphthene earlier, and then carry out the selectivity extracting with chloroform.This step can allow large scale purification, and need not seek help from conventional chromatography.
Further, derivative 11 can be in pyridine tribenzylization, with benzyl chloride and sodium hydride benzylization in dimethyl formamide, use acetolysis at last, to provide final ribitol derivative, the latter is by the distillation method purifying.Press following scheme with 15 ribosylation of ribitol derivative 14 usefulness ribofuranose peracetic acid esters.Behind deacetylation, by using a kind of new absorption and desorption method, can obtain triol 4 on a large scale, and chromatographic step that need not any routine.
Then, triol 4 is carried out the dibenzyl glycosylation reaction that the present invention found.Can obtain derivative 5 through this reaction, it is after column chromatography purification is handled and obtain.The allyl group of intermediate 5 tautomerizes to propenyl, imports phosphonic acid ester in the free position 3 of ribose units then.Remove propenyl, obtain crucial midbody derivant 19.Use similar approach, the hydroxyl of the position 3 of derivative 17 is carried out acetylizing, then the hydrolysis propenyl; 5 import phosphonic acid ester in the position, and to the deacetylated effect of product, make crucial disaccharides 23 on position 5 phosphonic acid ester be arranged, and on position 3 free hydroxyl group are arranged.
All form the structure of product and pass through nucleus magnetic resonance
1H and
13C and H-H and H-X related experiment confirm.Purity is by tlc or HPLC check.
From derivative 18 or 22, can obtain derivative 24 and 25, the latter also as the receptor of polycondensation, will show this point.
Studied the oligomerization reaction under different conditions, reaction is always only based on three compositions: crucial disaccharides (19 or its analogue 23); The promoter of reaction, it can be pivalyl chloride, diamantane 1-carbonyl chloride or the sterically hindered chloride of acid of other tool; And the 3rd composition, but its quencher reaction, and import spacerarm simultaneously.This composition position endways contains functional activation group or its precursor, for example sees 24,25 or 26.
As for spacerarm, under 24,25 or 26 situation 5-nitrine-3-oxa-amylalcohol.Spacerarm can use has general formula R
1Arbitrary compound of-Y-OH, wherein Y is the spacerarm chain, it can be aliphatic chain.Aliphatic chain can comprise insertion fragrant chain wherein or the heteroatoms number of swinging between 0-5.R
1Be the functional group of spacerarm terminal position, it can be NH
2, COOR, CHO, SH or arbitrary their precursor.
Disclosed the top condition of some situations being carried out polycondensation, for example in methylene dichloride-pyridine, disaccharides 19, spacerarm 5-nitrine-3-oxa-amylalcohol 26 and pivalyl chloride reaction draw product.Can obtain to contain the part of oligomer 27, carry out the LH-20 gel chromatography after the oxidizing reaction and with the LH-20 in the methyl alcohol after, be 70-85% based on the productive rate of disaccharides.
Oligosaccharide mixture 27 is when draping over one's shoulders palladium wood carbon and exist, and hydrogenation in methyl alcohol-ethyl acetate-water-acetate obtains thick oligosaccharide mixture 28.If spacer groups needs activation, this process is carried out better at next step.For example, in dimethyl formamide, the N-hydroxy-succinamide ester that will belong to β-maleimide propionic acid adds oligomer 28.After reaction finishes, use distilled water diluting gained solution, and depressed 1000 cutoff films and diafiltration at nitrogen.The product 30 of Huo Deing is the activatory oligosaccharide mixture like this, can be used for coupling processing.
In all situations, the structure that forms product is passed through nucleus magnetic resonance
1H and
13C and H-H and H-X related experiment confirm.
Use the propane thioic acid derived protein, it shelters mercaptan into disulphide by importing.To this derivatization reaction, available reagent such as SPDP or DSP react under nitrogen environment with dithiothreitol (DTT) then.For Neisseria meningitidis (Neisseria meningitidis) OMP or Toxoid,tetanus, excess reagent is with aqueous ethanol (20-95%) precipitation, and is centrifugal then and remove.In following scheme, set forth the processing that Neisseria meningitidis OMP is carried out.
Under inert environments, mercaptan protein is mixed with the activation oligosaccharides, the latter filters and lyophilize by 0.2 micron in advance.This reaction is by ethanol sedimentation quencher, centrifugal then or diafiltration.
In addition, can remove excessive activating reagent from conjugate by diafiltration.Twice separating treatment removed nearly all and the non-link coupled oligosaccharides of protein, makes the quality of final product very stable.
The mixture of oligosaccharides 30 can with other carrier coupling (for example lipid) coupling.Like this, for example when carbodiimide existed, with 2,33 reactions of 3-two-octadecyl oxygen base succinimide Succinic Acid propyl ester can obtain binding substances 34.
Can dilute synthetic oligosaccharide mixture and proteinic conjugate, or in the physiological buffer of capacity, rebuild, and can mix, to obtain final vaccine preparation with additive (as adjuvant, sanitas or other).
Equally, before preparation is handled or between, vaccine can be mixed with other vaccine, wherein said other vaccine is from used type in the immunization protocol to one-year-old following baby at present.For example, mix, can form the combined vaccine of anti--Hib and anti--meningitis b type with the outer membrane protein (OMP) of Neisseria meningitidis b type.After DTP mixes, can obtain to resist-Hib and diphtheria, Whooping cough and tetanic associating tetravalent vaccine.
Set forth the immunogenicity of the combined vaccine of oligosaccharides 30 and meningococcus outer membrane protein with some animal models.The existing of the ELISA method can detect this vaccine institute inductive anti--Hib capsular polysaccharide antibody (people such as D.C.Phipps, immunological method magazine (J.Immunolog.Methods), 1990.135.121-8).This results are shown in Figure 2-5.
For rabbit, when using the vaccine formulation of alumina-free, can induce higher reaction after first dose, but these two kinds of prepared products are identical at second dose of afterreaction.All can be observed high antibody titers under the both of these case.
In the example of Sprague's-Dao Li rat, the vaccine that is prepared by two kinds of different conjugates of carbohydrate-protein ratio also shows high anti--Hib antibody titers.
For the Balb/c mouse, from a kind of similar conjugate obtained vaccine-induced high anti--Hib capsular polysaccharide antibody titers.
The mixture of oligosaccharides 30 can with matrix coupling, for example polyacrylamide.Product can be used for detecting the anti--Hib antibody by the people of immunity or laboratory animal.Mixture also can with the latex coupling, and be used to detect ill or the people's that inoculated antibody.According to N.Bovin (the compounding sugar magazine, 1998,15,431-446), the activatory polyacrylamide can with oligosaccharides 30 mixture reactions.Ability to the polyacrylamide conjugate of HbO-HAS and 30 compares, the result as shown in Figure 6, wherein said HbO-HAS is the suggesting material that is used to detect anti--Hib antibody.The product that contains synthesis of oligose can reach reaction-noise ratio preferably.
Embodiment:
Embodiment 1: synthetic 5-O-allyl group-2,3-two-O-benzyl-D-ribitol 11
The methyl 2 of allylation-100g, 3-O-isopropylidene-D-ribofuranose is dissolved in the 70mL allyl bromide 98, in the presence of 75mL 50% sodium hydroxide aqueous solution and 2.6g season butyl ammonium iodide, stirs 12 hours.Stop then stirring, and carry out the separation of phase.Contain water (70mL) with dichloromethane extraction, the organic phase that dry then also volatilization merges.
Hydrolytic action-gained syrup is dissolved in 1.5L methyl alcohol.Add 3.6mL aqueous sulfuric acid (0.4N) to this solution, and mixture was refluxed 3 hours.After reaction finishes,, filter resulting salt and evaporating solns with the sodium bicarbonate neutralization.Use the ethyl acetate extraction resistates, dry and evaporation.Product was in vacuum-drying at least 2 hours.
Benzylation-products therefrom is dissolved in the 450mL dimethyl formamide.Gained solution is cooled to 0 ℃ and the slow 50g of adding sodium hydride.Stir this mixture and drip benzyl chloride (150mL) after 30 minutes.Stir after 2 hours, drip 20mL methyl alcohol to this reaction.In vacuum-evaporation gained suspension, be dissolved in methylene dichloride again and wash with water.With dried over sodium sulfate and evaporate organic phase.
Hydrolytic action-above-mentioned reaction gained syrup is dissolved in the 1.5L diox.Adding HCl (2N, 1.5L), and in 75-80 ℃ of this system of heating.Stopped reaction after 2 hours, and carry out the separation of phase.Contain water 2 times with the 200mL dichloromethane extraction, and the organic phase of evaporation merging.Enriched product is dissolved in methylene dichloride (1L), and water (400mL), saturated solution of sodium bicarbonate (300mL) and water (400mL) are washed successively, at last with dried over sodium sulfate and evaporation.
Reduction reaction-gained syrup is dissolved in ethanol (800mL), and with system cools to 20 ℃.Add 24g NaBH then
4Mixture is in stirring at room 1.5 hours, and after reaction finishes, with acetate at the excessive borohydride of pH destruction when pH is 7-8.Filtration is also evaporated this solution.Resistates is dissolved in the 500mL methylene dichloride, and this organic solution washes with water, dried over sodium sulfate, and is used for the following example.
Embodiment 2: purifying 5-O-allyl group-2,3-two-O-benzyl-D-ribitol 11
To the thick 5-O-allyl group-2 from the foregoing description, the dichloromethane solution of 3-two-O-benzyl-D-ribitol 11 adds 300g silica gel, and manual this mixture that stirs is adsorbed in solid phase until product.Vacuum-evaporation suspension is to remove methylene dichloride.The silica gel that contains product places percolator.With cyclohexane extract 48 hours, to remove impurity.To extract solvent replacement is that methylene dichloride is used for the product extraction, obtains chromatographically pure faint yellow syrup.Productive rate 75-95%.NMR
13C?δ60.7(C-1)，70.2(C-4)，71.0，71.7，72.0，73.6(PhCH
2C-5，OCH
2CH＝)，79.2，79.3(C-2，3)，117.1(CH
2＝)，127.5-128.2(Ph)，134.3(CH＝)，138.0，138.1(Cipso).
Embodiment 3: synthetic 5-O-allyl group-2,3,4-three-O-benzyl-D-ribitol 14 tribenzylizations (Trytilation)-from the 100g 5-O-allyl group-2 of the foregoing description, 3-two-O-benzyl-D-ribitol 11 vacuum-dryings is dissolved in the 600mL pyridine then.Add 75g chlorinated triphenyl methane and 0.5g dimethyl aminopyridine to this solution, and mixture was stirred 6 hours in 50 ℃.After reaction finishes, evaporating solvent, and resistates is dissolved in the 500mL methylene dichloride, this solution with water (1L) is washed, dry and evaporation.Resistates was in vacuum-drying 3 hours.
Benzylation-be dissolved in the 300mL dimethyl formamide from the syrup of above-mentioned reaction, and solution is cooled to 5 ℃.Slowly add sodium hydride (25g), continue then to stir 30 minutes.Slowly add benzyl chloride (40mL) then, and keep and stirred 1 hour.After reaction finishes, cooling once more, and slowly add 10mL methyl alcohol, to destroy excessive reagent.Evaporating solvent, and resistates is dissolved in the 500mL methylene dichloride, wash with 1L.Organic phase is also evaporated with dried over sodium sulfate.
Hydrolytic action-resistates is dissolved in acetate (1L) and 110mL water, and stirs reaction in 1.5 hours in 80 ℃.By the evaporative removal solvent, and resistates is dissolved in the 500mL cyclohexane.Organic phase is cooled to 0-5 ℃, 4 hours; Filter then, wash with water, dried over sodium sulfate and evaporation.By in 200-220 ℃ and 0.1mm distillation, obtain pure product.Based on 100g ribose, output is 80-90g.NMR
13C?δ61.2(C-1)，69.6，71.8，72.1，72.3.73.9(PhCH
2C-5，OCH
2CH＝)，78.0，78.8，78.9(C-2，3，4)，116.8(CH
2＝)，127.5-128.2(Ph)，134.7(CH＝)，138.0，138.1，138.2(Cipso).
Embodiment 4: synthetic 5-O-allyl group-2,3,4-three-O-benzyl-1-O-(β-D-ribofuranose)-D-ribitol 4
The product of glycosylation-the foregoing description (370g) is dissolved in the anhydrous ethylene dichloride of 2.7L, and is transferred to reactor.Solution is cooled to 0-25 ℃, adds opaque molecular sieve 4 (207g), after 15 minutes, slowly add boron trifluoride etherate (407mL) with 15mL/min.At last, in 20 minutes, slowly add D-ribofuranose peracetic acid ester 15 (370g) that are dissolved in anhydrous ethylene dichloride (1L).Stir reaction in 3 hours, and observe by the TLC (thin-layer chromatography) of hexane/ethyl acetate (2/1).Dull and stereotyped by using 5%H2SO4 (c) carbonization in ethanol.After reaction finishes, be 7 until pH, add saturated solution of sodium bicarbonate (800mL) then, and continue to stir 30 minutes with triethylamine (222mL) neutralization.The pH of reaction must remain on neutrality.The vacuum filtration reactor content.Solid is washed 2 times with ethylene dichloride (200mL), to extract any residual product in the solid.Discard solid phase, and with 600mL water extraction organic phase 2 times, with dried over sodium sulfate and vacuum-evaporation.
The syrup of deacetylation-above-mentioned reaction gained is dissolved in methyl alcohol (2L), adds the methanol solution (1%) with sodium methylate, reaches 9 until pH.Continue nearly 2 hours of reaction.After reaction finishes,, reach 6-7 until pH with the acidic resins neutralization.Resin is removed in vacuum filtration.Syrup (427g) contains target product and 5-O-allyl group-2,3,4-three-O-benzyl-D-ribitol 4, D-ribose and other undetermined impurity.
Embodiment 5: purifying 5-O-allyl group-2,3,4-three-O-benzyl-1-O-(β-D-ribofuranose)-D-ribitol 4
The syrup of the foregoing description is dissolved in methylene dichloride (1L).In solution, add silica gel (1kg) and manual this mixture that stirs, be adsorbed in solid phase until product.Vacuum-evaporation suspension is to remove methylene dichloride.Gained solid vacuum-drying 2 hours is to remove the methylene dichloride of any trace.
The silica gel that contains product places percolator.Removed impurity in 48 hours with cyclohexane extract.To extract solvent replacement is chloroform, extracts product, obtains faint yellow syrup.Output 238g.NMR?
1H?δ5.85(m，1H，CH＝)，5.20(m，2H，CH
2＝)，4.85(s，1H，H-1′)，
13C?δ62.8(C-5’)，77.7，77.9，78.2(C-2，3，4)，84.0(C-4’)，107.2(C-1’)。
Embodiment 6: synthetic 5-O-allyl group-2,3,4-three-O-benzyl-1-O-(β-D-2 ', 5 '-two-O-benzyl-ribofuranose)-D-ribitol 5
The compound (200g) of benzylization-the foregoing description gained is dissolved in toluene (2L).Add Bu to this solution
2SnO (80g), and with mixture backflow 4 hours.Room temperature adds NaH 50% (56g) with small portion, and stirs the mixture 30 minutes in 80 ℃.Add season butyl ammonium iodide (62g), restir mixture 1 hour.Add benzyl chloride (148mL) then, and continue stirring reaction a few hours in 80 ℃.Repeated similarly to add benzyl chloride every 30 minutes, show that until TLC (hexane/ethyl acetate-2/1) primary product exists with the disaccharides of dibenzylization.Cooling reaction also neutralizes with the methanol solution of 1%HCl.React through diatomite filtration reduction vaporization then.The product that contains some salt of gained is dissolved in ethyl acetate, and filtration under diminished pressure also concentrates.Raw sugar slurry toluene-acetone 60/1 solvent systems column chromatography purification.Purifying thing 5 obtains with syrup (100g) form.NMR-
1H?δ5.96(m，1H，-CH＝)，5.18(m，2H，CH
2＝)，5.02(s，1H，H-1)
Embodiment 7: Synthetic 2, and 3,4-three-O-benzyl-1-O-(β-D-2 ', 5 '-two-O-benzyl-3 '-O-triethylamine phosphonic acid ester-ribofuranose)-D-ribitol (19)
Allylic isomerization-in high vacuum was with dry 2 hours of the foregoing description gained syrup 20g.Syrup is dissolved in anhydrous dimethyl sulfoxide (100mL), and adds potassium tert.-butoxide (6.4g).In 100 ℃ of stirrings reaction in 1 hour, add the 250mL frozen water then.Dropwise being added dropwise to concentrated hydrochloric acid makes pH reach 7.Extract mixture 3 times with diethyl ether 80mL.Merge organic phase, with dried over sodium sulfate and evaporation.
Phosphonic acids turns usefulness-anhydrous acetonitrile (34mL) solution of imidazoles (1.5g) is cooled to 0 ℃ into.Add phosphorus trichloride (0.56mL) and triethylamine (3.1mL).Gained solution stirring 15 minutes.Disaccharides from above-mentioned reaction adds to this mixture that is dissolved in anhydrous acetonitrile (3mL).The gained mixture is in stirring at room 15 minutes, and by adding the three second amine bromide termination reactions of 1M.Continue to stir 10 minutes, add methylene dichloride, and carry out the separation of phase.Organic phase with three cold second Ammonium bicarbonate food grade solution wash, dry and evaporation.
Hydrolytic action-the product of propenyl is dissolved in 60% acetate, and stirs 30 minutes in 70 ℃.Evaporative removal solvent, and product is dissolved in methylene dichloride, with three second Ammonium bicarbonate food grades wash, dry and evaporation.Products therefrom is carried out column chromatography, obtained pure compound, productive rate is between 70-85%.NMR?
1H?δ6.85(d，H-P)，4.95(s，H-1)，4.60(m，H-3)，2.93(q，NCH
2CH
3)1.20(t，NCH
2CH
3).
13C?δ105.9(C-1).
Polycondensation between embodiment 8:19 and 26 (ratio 10-1)
(10-1, the solution of compound 19 (1g) 1mL) adds trimethyl-acetyl chloride (0.14mL), stirring reaction 20 minutes to being dissolved in pyridine-triethylamine.Add spacerarm 26 (29.2mg) and new trimethyl-acetyl chloride (0.9mL), and stirring reaction 1 hour.Add and be dissolved in pyridine-water (7.3mL; I 20-1)
2(1.1g) solution, stirring reaction 30 minutes.Mixture dilutes with methylene dichloride, washes with hypo solution (1M) and cold three second amine bromide (0.5M) solution, then with dried over sodium sulfate and evaporation.Products therefrom is dissolved in methyl alcohol, and carries out chromatography with same solvent in dextran LH-20 post.Merge the fraction and the evaporation that contain oligomer.Productive rate 80%.
Polycondensation between embodiment 9:19 and 26 (ratio 10-1)
(10-1, compound 19 (1g) 1mL) and the solution of spacerarm 24 (14.6mg) add trimethyl-acetyl chloride (0.23mL), and stirring reaction 2 hours to being dissolved in pyridine-triethylamine.Add and be dissolved in pyridine-water (7.3mL; I 20-1)
2(1.1g) solution, product is handled with mode similar to Example 8.
Polycondensation between embodiment 10:19 and 26 (ratio 5-1)
(10-1, compound 19 (1g) 1mL) and the solution of spacerarm 24 (29.2mg) add trimethyl-acetyl chloride (0.23mL), and stirring reaction 2 hours to being dissolved in pyridine-triethylamine.Add and be dissolved in pyridine-water (7.3mL; I 20-1)
2(1.1g) solution, product is handled with mode similar to Example 8.
Polycondensation between embodiment 11:23 and 26 (ratio 5-1, solvent pyridine)
Add trimethyl-acetyl chloride (0.23mL) to the solution of compound 23 (1g) that is dissolved in pyridine (1mL) and spacerarm 26 (29.2mg), and stirring reaction 2 hours.Add and be dissolved in pyridine-water (7.3mL; I 20-1)
2(1.1g) solution, product is handled with mode similar to Example 8.
Polycondensation between embodiment 12:19 and 24 (ratio 5-1, solvent pyridine)
Solution to compound 19 (1g) that is dissolved in pyridine (1mL) and spacerarm 24 (29.2mg) adds trimethyl-acetyl chloride (0.23mL), and stirs reaction in 2 hours.Add and be dissolved in pyridine-water (7.3mL; I 20-1)
2(1.1g) solution, product is handled with mode similar to Example 8.
Embodiment 13: from the hydrogenation of the product of embodiment 8-12
Crude product to the foregoing description 8-12 carries out hydrogenization, 10% drape over one's shoulders on the palladium wood carbon and react in the mixed solution of ethyl acetate, alcohol and water-acetate (1-2-2-0.1).At last, product carries out the ion-exchange chromatography purifying in dextran C-25Na.Characterize cryodesiccated product by NMR, and shown the Rib-Rib-of basic repeating unit phosphoric acid and spacerarm.After percolation process and the Treatment with Ultrafiltration, obtained the activation fraction,, passed through the film of 10000 cutoffs at first by the film of 1000 cutoffs.The solution and the lyophilize of merga pass 10000 films obtain finally 28, and according to the difference of reaction conditions, overall yield is between the 20-80% based on initial disaccharides.NMR
1H δ 5.12 (H-1), 4.60 (H-3), 3.29 (CH
2NH
2).
31P δ 2.14 (spac-P-Rib) 0.74 (ribitol-P-Rib).
Embodiment 14. synthesis of derivatives 5-(3-maleimide propionic acid amide)-3-oxa-pentyl oligomerization ribosyl (ribosil) ribitol phosphoric acid ester 30
Distilled water (0.1mL) solution to the foregoing description (3.34mg, 1.73 μ mol) adds 0.7mg (2.62 μ mol) N-hydroxy-succinamide base β-maleimide propionic ester 27, and the latter is dissolved in N, dinethylformamide (0.4mL).React after 4 hours, solution is resuspended in distilled water (0.5mL) and centrifugal (10 minutes, 3500 rev/mins) in vacuum-evaporation.Dilute with water supernatant, and the Amicon equipment ultrafiltration of the film of apparatus 1000 cutoffs.The lyophilize retentate.Productive rate is between 85 to 95%.NMR：
1H，δ6.95(s，2H，CH＝CH)，5.01(s，H-1)3.41(t，2H，CH
2NH)，2.56(t，2H，CH
2a).
Embodiment 15: available from the product of embodiment 13 by the ion exchange chromatography analysis
The hydrogenant of embodiment 8-12 and as the product of sodium salt by HR5/5mono Q post, with the sodium-chlor linear gradient carry out ion exchange chromatography analyze (P.Constantino, ycol., vaccine 1999,17,1251-1263).The elution chromatography curve of embodiment 8 is seen Figure 1B, and has shown the fragment of different sizes.As with the result of they and bibliographical information and Figure 1A relatively, the former representative has the chromatogram of pentamer, can reach a conclusion thus: the fragment that presents in the mixture from 4-5 repeating unit to above 20 repeating units.
Embodiment 16: with the coupling of Neisseria meningitidis outer membrane protein
400mg Neisseria meningitidis outer membrane protein composite (OMPC) is dissolved in the PBS damping fluid of 80mL pH8, and the latter uses N in advance
2(g) lavation.Solution N
2(g) lavation is 5 minutes, stirs in ice-water bath simultaneously.Add the 1.6mL DSP solution that is dissolved in the dimethyl formamide, and mixture was stirred 2 hours gently in 4 ℃.Adding is dissolved in the 1.6mL DTT solution of PBS, and continues at 4 ℃ of stirrings 1 hour.The suspension of gained is transferred to and contains the centrifugal flask of the cold alcoholic acid of 20-400mL.In 1800 rev/mins 4 ℃ centrifugal flasks 30 minutes, supernatant discarded.Add new ethanol to solid, after adding 200-400mL ethanol, repeated centrifugation step once more.The resuspended precipitation of PBS damping fluid with 80mL pH7-9.In gained solution, add synthetic oligosaccharides 30, and continue to stir 1-48 hour.In case processing finishes, behind 30000 cutoff diafiltrations, repeat ethanol sedimentation-centrifugally operated.Rebuild retentate with the PBS damping fluid, making concentration is Hib 40-80 μ g/mL.
Embodiment 17: with combining of Toxoid,tetanus
The concentration of pH8 PBS preparation is the Toxoid,tetanus solution N of 5mg/mL
2(g) air-blowing is 5 minutes.Maintain when stirring in the ice-water bath, add the 1.6mL DSP solution that is dissolved in the dimethyl formamide, and mixture was stirred 2 hours gently in 4 ℃.Adding is dissolved in the 1.6mL DTT solution of PBS, and continues at 4 ℃ of stirrings 1 hour.Gained solution is transferred to the ultrafiltration apparatus of tool 10000 cutoff films.Add and use N in advance
2(g) air-blowing fresh buffer, to the solution diafiltration several times, negative until solution E lman test by film.In gained solution, add synthetic oligosaccharides 30, and continue to stir 1-48 hour.After processing finishes,, negative to the phenol-sulphur test of sugar until solution by film once more to the solution diafiltration.Rebuild solution with the PBS damping fluid at last, making concentration is Hib 40-80 μ g/mL.
Embodiment 18: with the coupling of two octadecyl glyceryl hemisuccinic acid esters.
The product of embodiment 8 (10mg) is dissolved in the 1mL dimethyl formamide, and adds to two octadecyl glycerine hemisuccinic acid ester solutions, and the latter is a N-hydroxy-succinamide ester 31.Add ethyl diamines propyl group carbodiimide (5mg) to this solution, stir reaction in 6 hours.Add new carbodiimide, continue to stir 6 hours.Evaporating solvent also adds to C18-silicagel column (1g) with mixture.Wash with water and slough except that oligosaccharides.Methanol-water eluted product with gradient concentration.Productive rate is 85%.NMR?
1H?δ5.12(H-1)，4.60(H-3)，3.40(CH
2NH)，1.30(CH
2)，0.90(CH
3)。
Embodiment 19: the vaccine of the no adjuvant of preparation
Be dissolved in phosphoric acid buffer, concentration is the immunogen of the embodiment 16 of 40 μ g/mL, under aseptic condition, dilute with distilled water in 4-8 ℃.Stirred suspension 10 minutes.The final concentration of Hib antigen is by determining the estimation of ribose and gross protein, and can reset by adding more damping fluid, is 20mg/mL until the antigenic final concentration of Hib.Add Tiomersal to final concentration be 0.1-0.001%.Gained suspension is the final volume of the anti-Hib combined vaccine of no adjuvant.
Embodiment 20: preparation is with the anti-Hib vaccine of aluminum oxide as adjuvant
Be dissolved in phosphoric acid buffer, concentration is the immunogen of the embodiment 16 of 40 μ g/mL, under aseptic condition, mix with isopyknic, as to be dissolved in distilled water 1-0.01mg/mL aluminum oxide in 4-6 ℃.Keep stirring 20 minutes in 4-8 ℃.The final concentration of Hib is by determining the estimation of ribose and gross protein, and can reset by adding more damping fluid, is 20mg/mL until the antigenic final concentration of Hib.Add Tiomersal to final concentration be 0.1-0.001%.Gained suspension is the final volume of the anti-Hib combined vaccine of aluminum oxide.
Embodiment 21: the combined vaccine for preparing anti--Hib and meningococcemia B
Be dissolved in phosphoric acid buffer, concentration is the immunogen of the embodiment 16 of 80 μ g/mL, mix with the Neisseria meningitidis Type B outer membrane protein complex body (OMPC) of certain volume in 4-6 ℃ under aseptic condition, the concentration that the latter is used for VAMEMGOC BC at present is 100-200mg/mL PBS.After soft magnetism power stirred 20 minutes homogenizations, reactant mixed with isopyknic, as to be dissolved in distilled water 1-0.01mg/mL aluminum oxide.Keep stirring 20 minutes in 4-6 ℃.The antigenic final concentration of Hib passes through the Lowry method to be determined the estimation of ribose and gross protein, and can reset by adding more damping fluid, is 20mg/mL until the antigenic final concentration of Hib.Add Tiomersal to final concentration be 0.1-0.001%.Gained suspension is the final volume of uniting anti-Hib and meningococcemia B in the aluminum oxide.
Embodiment 22: the combined vaccine for preparing anti-Hib and anti--DTP
Be dissolved in phosphoric acid buffer, concentration is the immunogen of the embodiment 16 of 80 μ g/mL, under aseptic condition, mix in 4-6 ℃ of DTP with the 4x concentration of certain volume.After soft magnetism power stirred 20 minutes homogenizations, reactant mixed with isopyknic, as to be dissolved in distilled water 1-0.01mg/mL aluminum oxide.Keep stirring 20 minutes in 4-6 ℃.The antigenic final concentration of Hib passes through the Lowry method to be determined the estimation of ribose and gross protein, and can reset by adding more damping fluid, is 20mg/mL until the antigenic final concentration of Hib.Add Tiomersal to final concentration be 0.1-0.001%.Gained suspension is the final volume of anti-Hib of uniting of aluminum oxide and anti--DTP.
Embodiment 23: by the immunoassay of the vaccine of synthetic oligosaccharide mixture and OMP preparation
Use indirect elisa method, measure antibody response as envelope antigen with Hib-HAS.The result is shown in Fig. 2-5.
Embodiment 24: the coupling of synthetic oligosaccharide mixture and p-nitrophenyl acrylate
Oligosaccharide mixture (10mg) from embodiment 13 is dissolved in dimethyl formamide, and adds to nitrophenyl acrylate (10-30mg) solution (1mL) that is dissolved in dimethyl formamide.Add the 0.1-0.5mL triethylamine, kept stirring reaction 16 hours.Add 0.1-0.5mL ammoniacal liquor, continue to stir 24 hours.The sephadex LH-20 post of acetonitrile on the solution.Carry out wash-out with acetonitrile-water.The oreinol diphenol is analyzed ribose, merges positive fraction and lyophilize.Productive rate is usually above 80%.
Embodiment 25: the synthesis of oligose conjugate detects the ability of anti--Hib antibody
Be dissolved in 96 orifice plates carbonate-bicarbonate buffer, add half with 1-100 μ g/mL concentration from the product solution of the foregoing description.Second half of plate adds the antigen HbO-HAS of recommended density.The mice serum of crossing with vaccine immunity carries out ELISA mensuration, and wherein said vaccine contains and the natural capsular polysaccharide of Toxoid,tetanus link coupled.The result who obtains as shown in Figure 6.
The accompanying drawing summary:
Fig. 1. the typical layers of the oligomer level branch that is obtained by embodiment 15 is analysed spectrum, and it is for by Mono Q HR 5/5, the ion exchange chromatography that carries out with NaCl 0-500mM linear gradient, wherein A-pentamer; B-is from the oligomer of embodiment 10.
Fig. 2. the antibody response in the rabbit of the vaccine immunity of usefulness embodiment 16.
Fig. 3. the antibody response in the rabbit of the vaccine immunity of usefulness embodiment 15.
Fig. 4. the antibody response in the rat of the vaccine immunity of usefulness embodiment 15.
Fig. 5. the antibody response in the Balb C mouse of the vaccine immunity of usefulness embodiment 15.
Fig. 6. the 26-PAA conjugate detects the ability of anti--Hib antibody.
Claims (21)
1. oligosaccharide mixture, it comprises the repeating unit of formula for (phosphoric acid-ribose-ribitol) n or (ribose-ribitol-phosphoric acid) n, and wherein this oligosaccharide mixture obtains by synthetic, comprises at least 5 compounds with structure A or B, n is a value between 4 and 25, and R wherein
1Or R
2Be to be used for and carrier-bound spacerarm, its condition is: if R
2=H, then R
1If=spacerarm is or R
1=H, then R
2=spacerarm.
2. according to the oligosaccharide mixture of claim 1, wherein having formula is that this compound of A or B has only the n difference to each other for (phosphoric acid-ribose-ribitol) n or (ribose-ribitol-phosphoric acid) n, structure, and wherein n is a value between 4 and 25.
3. according to the oligosaccharide mixture of claim 3, wherein spacerarm refers to arbitrary aliphatic hydrocarbon chain, and it contains 0-5 heteroatoms and/or aromatic series chain; Always there is the NH of functional group in the other end at spacerarm
2, COOR, CHO, SH or their precursor, it can form chemical bond with the molecule as carrier.
4. the method for synthesis of oligose mixture, wherein said oligosaccharide mixture comprises the repeating unit of formula for (phosphoric acid-ribose-ribitol) n or (ribose-ribitol-phosphoric acid) n, wherein in basic solvent and spacerarm when participating in reaction, two sugar derivativess 19 or 23 are carried out one-step polycondensation reaction in the presence of promoter, wherein promoter can for and activate the sterically hindered acid derivant of tool, but spacerarm is as the growth of acceptor quencher oligomer chain.
5. according to the method for claim 4, wherein disaccharides intermediate 19 or 23 and the mol ratio of promoter be 1/1-3.
6. according to the step of claim 4 and 5, wherein promoter is the activated derivatives of ester sterically hindered acid.
7. according to the method for claim 4 and 6, the activated derivatives of the preferred pivalyl chloride of promoter, diamantane carbonyl chloride or phosphoric acid wherein.
8. according to the method for claim 4, wherein disaccharides intermediate 19 or 23 and the mol ratio of spacerarm be 5-10/1.
9. according to the method for claim 4-8, wherein disaccharides is 19, and spacerarm is 24, and promoter is a pivalyl chloride, and solvent is a pyridine.
10. the method for synthesizing the oligosaccharide mixture of claim 1-3; wherein the compound that the method according to claim 4-9 is obtained carries out the oxidation of further phosphonic acids to phosphoric acid; remove the benzyl protection group by hydrogenization then; remove protection or activation spacerarm, and handle by a step or a few step molecular sieve and to remove less than 4 or greater than the fraction of 25 repeating units.
11. comprise the immunogen of the oligosaccharide mixture of claim 1-3, wherein this oligosaccharide mixture combines with the carrier molecule that is selected from protein, peptide or lipid.
12. be used to prevent the vaccine of the infectious diseases that causes by Haemophilus influenzae b type, its comprise with or not with the immunogen of the claim 11 of adjuvant or the preparation of other additive.
13. according to the vaccine of claim 12, itself and other vaccine such as resistance of hepatitis B, anti--meningococcus A, B and/or C, DPT, poliomyelitis, anti-streptococcus pneumoniae 1,5,6B, 6A, 14,19F, 19
aAnd/or 23F, unite use, or unite use with their immunogens separately.
14. synthetic pure disaccharides intermediate 2,3, the method of 4-three-O-benzyl-5-O-allyl group-(β-D-ribofuranose)-D-ribitol 4, it is by 2,3, the ribosylation of 4-three-O-benzyl-5-O-allyl group-D-ribitol 14 and ribofuranose peracetic acid ester, deacetylation then, get compound 4, it is used silica gel adsorption-desorption purifying.
15. the method for synthetic disaccharides intermediate 17, it comprise with disaccharides 4 and Dibutyltin oxide, season butyl ammonium iodide, sodium hydride and benzyl chloride with 1: 0.5-2: 0.001-1,0.5-10: the ratio of 1-5 is as the dibenzyl processing in the solvent of toluene, benzene, tetrahydrofuran (THF) or dimethylbenzene, obtain having the compound of formula 5, allyl group is isomerizated into and is propenyl then.
16. disaccharides 5-O-propenyl-2,3,4-three-O-benzyl-1-O-(2 ', 5 '-two-O-benzyl-β-D-ribofuranose)-D-ribitol 17 as intermediate, are used for the synthetic of derivative 19 and 23.
17. two sugar derivativess 2,3,4-three-O-benzyl-1-O-(2 ', 5 '-two-O-benzyl β-D-ribofuranose)-D-ribitol-5-O-triethylamine phosphonic acids 23 and 2,3,4-three-O-benzyl-1-O-(2 ', 5 '-two-O-benzyl-β-D-ribofuranose)-D-ribitol-3 '-O-triethylamine phosphonic acids 19 is used for the oligomerization process of claim 5-7 as crucial intermediate.
18. prepare the method for disaccharides 19 or 23 by disaccharides 17, it comprises with phosphorus trichloride-imidazoles carry out phosphonic acidsization, then after hydrolysis propenyl or the acetylizing, and the hydrolysis propenyl, and carry out phosphonic acids with the phosphorus trichloride imidazoles and turn usefulness into, deacetylated then.
19. the purposes of disaccharides 19 or 23, it is used for oligosaccharide mixture synthetic of claim 1-3.
20. comprise the macromole of the oligosaccharide mixture of claim 1-3, wherein oligosaccharide mixture combines with the immunologic inertia polymer materials.
21. the macromolecular purposes of claim 20, it is used to detect and quantitatively at the antibody of the capsular polysaccharide of Haemophilus influenzae b type.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CU121/1999 | 1999-08-30 | ||
| CU121/99 | 1999-08-30 | ||
| CU1999121A CU22904A1 (en) | 1999-08-30 | 1999-08-30 | OLIGOSACÁRIDOS DERIVED FROM RIBOSA- RIBITOL-PHOSPHATE, METHODS TO PREPARE THEM, IMMUNOGENS THAT UNDERSTAND THEM AND VACCINES THAT UNDERSTAND SUCH IMMUNOGENS |
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| Publication Number | Publication Date |
|---|---|
| CN1376158A true CN1376158A (en) | 2002-10-23 |
| CN1205217C CN1205217C (en) | 2005-06-08 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB008134731A Expired - Lifetime CN1205217C (en) | 1999-08-30 | 2000-08-15 | Oligosaccharides derived from ribose-ribitol-phosphate, and vaccines containing them |
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| Country | Link |
|---|---|
| US (1) | US6765091B1 (en) |
| EP (1) | EP1212334B1 (en) |
| JP (1) | JP4850377B2 (en) |
| KR (1) | KR100818163B1 (en) |
| CN (1) | CN1205217C (en) |
| AR (1) | AR025435A1 (en) |
| AT (1) | ATE308551T1 (en) |
| AU (1) | AU780816B2 (en) |
| BR (1) | BRPI0013686B8 (en) |
| CA (1) | CA2382602C (en) |
| CO (1) | CO5180646A1 (en) |
| CU (1) | CU22904A1 (en) |
| CZ (1) | CZ2002721A3 (en) |
| DE (1) | DE60023722T2 (en) |
| DK (1) | DK1212334T3 (en) |
| EA (1) | EA005381B1 (en) |
| ES (1) | ES2252045T3 (en) |
| HK (1) | HK1050693A1 (en) |
| MX (1) | MXPA02002066A (en) |
| NZ (1) | NZ517464A (en) |
| SI (1) | SI1212334T1 (en) |
| UA (1) | UA75579C2 (en) |
| WO (1) | WO2001016146A1 (en) |
| ZA (1) | ZA200201667B (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2005209303A1 (en) * | 2004-01-29 | 2005-08-11 | Biosynexus, Inc. | Use of amino-oxy functional groups in the preparation of vaccines conjugates |
| US8647621B2 (en) | 2009-07-27 | 2014-02-11 | Fina Biosolutions, Llc | Method of producing protein-carbohydrate vaccines reduced in free carbohydrate |
| JP6166533B2 (en) | 2009-07-31 | 2017-07-19 | ナショナル リサーチ カウンシル オブ カナダ | Outer shell epitope of H. pylori lipopolysaccharide |
| CA2836140C (en) | 2009-12-17 | 2016-06-21 | Fina Biosolutions, Llc | Chemical reagents for the activation of polysaccharides in the preparation of conjugate vaccines |
| ES2743156T3 (en) * | 2010-04-23 | 2020-02-18 | Serum Institute Of India Pvt Ltd | Simple method for simultaneous removal of multiple impurities from culture supernatants at ultra low levels |
| KR102549847B1 (en) | 2016-07-28 | 2023-06-29 | 막스-플랑크-게젤샤프트 츄어 푀르더룽 데어 비쎈샤프텐 에.파우. | Stable, hydrolysis-resistant synthetic polyribosylbitolphosphate derivatives as vaccines against Haemophilus influenzae type b |
| BR112020018974A2 (en) * | 2018-03-23 | 2021-01-05 | Koranex Capital | PRECISION GLYCOCONJUGATES AS THERAPEUTIC TOOLS |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| NZ223009A (en) * | 1986-12-31 | 1990-06-26 | Nl Rivm Of Thoven | Oligosaccharides containing d-ribose d-ribitol and phosphate units mimicing haemophilus influenzae type b antigens |
| EP0320942A3 (en) * | 1987-12-18 | 1989-10-04 | American Cyanamid Company | Novel polysaccharides novel macromolecular conjugates of the polysaccharides |
| US5843463A (en) * | 1990-12-21 | 1998-12-01 | Antexbiologics, Inc. | Adhesin-oligosaccharide conjugate vaccine for Haemophilus influenzae |
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1999
- 1999-08-30 CU CU1999121A patent/CU22904A1/en unknown
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2000
- 2000-08-15 US US10/070,101 patent/US6765091B1/en not_active Expired - Lifetime
- 2000-08-15 EA EA200200314A patent/EA005381B1/en not_active IP Right Cessation
- 2000-08-15 DK DK00956040T patent/DK1212334T3/en active
- 2000-08-15 AU AU68173/00A patent/AU780816B2/en not_active Ceased
- 2000-08-15 NZ NZ517464A patent/NZ517464A/en not_active IP Right Cessation
- 2000-08-15 BR BRPI0013686A patent/BRPI0013686B8/en not_active IP Right Cessation
- 2000-08-15 ES ES00956040T patent/ES2252045T3/en not_active Expired - Lifetime
- 2000-08-15 WO PCT/CU2000/000003 patent/WO2001016146A1/en active IP Right Grant
- 2000-08-15 CA CA2382602A patent/CA2382602C/en not_active Expired - Fee Related
- 2000-08-15 AT AT00956040T patent/ATE308551T1/en active
- 2000-08-15 EP EP00956040A patent/EP1212334B1/en not_active Expired - Lifetime
- 2000-08-15 UA UA2002021613A patent/UA75579C2/en unknown
- 2000-08-15 CZ CZ2002721A patent/CZ2002721A3/en unknown
- 2000-08-15 JP JP2001519710A patent/JP4850377B2/en not_active Expired - Fee Related
- 2000-08-15 SI SI200030780T patent/SI1212334T1/en unknown
- 2000-08-15 KR KR1020027002706A patent/KR100818163B1/en not_active IP Right Cessation
- 2000-08-15 CN CNB008134731A patent/CN1205217C/en not_active Expired - Lifetime
- 2000-08-15 MX MXPA02002066A patent/MXPA02002066A/en active IP Right Grant
- 2000-08-15 DE DE60023722T patent/DE60023722T2/en not_active Expired - Lifetime
- 2000-08-28 CO CO00064095A patent/CO5180646A1/en active IP Right Grant
- 2000-08-29 AR ARP000104496A patent/AR025435A1/en not_active Application Discontinuation
-
2002
- 2002-02-27 ZA ZA200201667A patent/ZA200201667B/en unknown
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2003
- 2003-04-23 HK HK03102864A patent/HK1050693A1/en unknown
Also Published As
| Publication number | Publication date |
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| BRPI0013686B8 (en) | 2021-05-25 |
| KR20020068501A (en) | 2002-08-27 |
| BRPI0013686B1 (en) | 2016-07-26 |
| CA2382602A1 (en) | 2001-03-08 |
| EA005381B1 (en) | 2005-02-24 |
| ATE308551T1 (en) | 2005-11-15 |
| DK1212334T3 (en) | 2006-03-20 |
| NZ517464A (en) | 2004-03-26 |
| ES2252045T3 (en) | 2006-05-16 |
| DE60023722T2 (en) | 2006-07-20 |
| BR0013686A (en) | 2002-08-13 |
| CA2382602C (en) | 2010-02-16 |
| DE60023722D1 (en) | 2005-12-08 |
| UA75579C2 (en) | 2006-05-15 |
| EP1212334A1 (en) | 2002-06-12 |
| EA200200314A1 (en) | 2002-10-31 |
| CZ2002721A3 (en) | 2002-07-17 |
| ZA200201667B (en) | 2003-08-27 |
| AR025435A1 (en) | 2002-11-27 |
| EP1212334B1 (en) | 2005-11-02 |
| AU6817300A (en) | 2001-03-26 |
| AU780816B2 (en) | 2005-04-21 |
| JP4850377B2 (en) | 2012-01-11 |
| MXPA02002066A (en) | 2004-07-30 |
| CU22904A1 (en) | 2004-01-23 |
| JP2003528032A (en) | 2003-09-24 |
| KR100818163B1 (en) | 2008-03-31 |
| HK1050693A1 (en) | 2003-07-04 |
| SI1212334T1 (en) | 2006-04-30 |
| US6765091B1 (en) | 2004-07-20 |
| WO2001016146A1 (en) | 2001-03-08 |
| CN1205217C (en) | 2005-06-08 |
| CO5180646A1 (en) | 2002-07-30 |
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